Integrin targeted imaging agents

ABSTRACT

Emulsions preferably of nanoparticles formed from high boiling liquid perfluorochemical substances, said particles coated with a lipid/surfactant coating are made specific to regions of activated endothelial cells by coupling said nanoparticles to a ligand specific for α v β 3  integrin, other than an antibody. The nanoparticles may further include biologically active agents, radionuclides, or other imaging agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/305,416 filed 16Dec. 2005 and now allowed which is a divisional of U.S. Ser. No.10/351,463 filed 24 Jan. 2003 now U.S. Pat. No. 7,255,875 which claimsbenefit under 35 U.S.C. § 119(e) to provisional application No.60/351,390 filed 24 Jan. 2002. The contents of these applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to nanoparticle-based emulsions that arespecifically targeted to integrins employing α_(v)β₃-specific targetingagents. More specifically, the invention relates to the use ofnon-antibody based compositions for such targeting.

BACKGROUND ART

The value of nanoparticulate compositions composed of perfluorocarbonnanoparticles coated with a surfactant layer to facilitate binding ofdesired components for imaging of various types is well established.See, for example, U.S. Pat. Nos. 5,690,907; 5,780,010; 5,989,520;5,958,371; and PCT publication WO 02/060524, the contents of which areincorporated herein by reference. These documents describe emulsions ofperfluorocarbon nanoparticles that are coupled to various targetingagents and to desired components, such as MRI imaging agents,radionuclides, and/or bioactive agents. Other compositions that havebeen used for targeted imaging include those disclosed in PCTpublications WO 99/58162; WO 00/35488; WO 00/35887; and WO 00/35492. Thecontents of these publications are also incorporated herein byreference.

The integrin α_(v)β₃, which binds to vitronectin, is recognized as amarker for neovasculature. It is relatively selective for activatedendothelial cells and essentially unexpressed on mature, quiescentcells. Based on this characteristic, it has been attempted to useantagonists to this integrin as anticancer agents. Kerr, J. S., et al.,Anticancer Res. (1999) 19:959-968 describe peptide mimetics which wereable to decrease neovasculature formation in a mouse model system. U.S.Pat. No. 6,153,628 describes 1,3,4-thiadiazoles and 1,3,4-oxadiazolesthat are α_(v)β₃ antagonists and are said to useful in the treatment ofdisorders related to angiogenesis, including inflammation, bonedegradation, tumors, metastases, thrombosis, and cell aggregationrelated conditions. U.S. Pat. Nos. 6,130,231 and 6,322,770 disclosefused heterocycles that are α_(v)β₃ antagonists and useful for the samepurposes, as does PCT publication WO 01/97848.

The WO 01/97848 publication discloses specific compounds that can belinked to ancillary substances, optionally through linker moieties,wherein these ancillary substances may include radionuclides, substancesuseful in magnetic resonance imaging, and X-ray contrast agents. Thispublication also discloses the use of these compounds coupled to certainultrasound contrast agents, typically containing gaseous bubbles.

In addition to its expression in activated endothelial cells, α_(v)β₃ isexpressed on vascular smooth muscle cells, including macrophage in thewalls of the vasculature. This complex binds cells to the surroundingmatrix and is thus employed by cells in the course of migration.Accordingly, α_(v)β₃ plays a role in restenosis by assisting themovement of cells into the lumen. A key component of restenosis involvesvascular smooth muscle cell activation, proliferation and migration.Integrin heterodimers, in particular the α_(v)β₃ integrin, arerecognized as critical elements in these processes by providing celladhesion to the extracellular matrix, inducing extracellularmetalloproteinase expression, and facilitating smooth muscle cellmigration. The α_(v)β₃ integrin is widely distributed among endothelialcells, stimulated monocytes, T-lymphocytes, fibroblasts, vascular smoothmuscle cells and platelets and binds to several extracellular matrixprotein ligands including osteopontin, vitronectin, thrombospondin, anddenatured collagens.

Antagonism of integrin mediated cell-matrix interactions within theballoon-stretched vessel walls inhibits inflammatory cell recruitment tothe injury site, limits smooth muscle cell proliferation and migration,and diminishes extracellular matrix protein synthesis. Selective andnonselective blockade of integrins with cyclic RGD peptide antagonistshave limited neointimal hyperplasia in several animal models ofrestenosis.

Restenosis is associated most often with angioplasty wherein, in anattempt to expand the vasculature using balloon catheters, thevasculature is broken, exposing the vascular smooth muscle cells. Theresulting fractures require the movement of cells into the lumen; theα_(v)β₃ acts to assist the migration through the matrix of collagen andfibrin to accomplish this. Accordingly, compositions that target α_(v)β₃may also be used to target smooth muscle cells and to image restenoses,in particular those associated with balloon angioplasty, and to deliveranti-proliferation agents such as paclitaxel, rapamycin, and othertherapeutic moieties such as radionuclides, small molecules, peptidesand nucleic acids.

While stent-based delivery systems offer the possibility of focaltherapeutic drug effects within the tunica media of arteries withoutincurring the adverse side effects of systemic drug administration, andproduce high local intimal concentrations of drug proximate to thestent-strut-arterial wall contact points, persistent highantiproliferative drug concentrations within the intima can impairarterial wall healing and reendothelialization, which promotesinflammation of the lumen lining and restenosis. The inventioncompositions avoid these problems.

It appears most peptidomimetics and neutralizing antibody α_(v)β₃antagonists have short half-lives and occupy the receptor for α_(v)β₃only transiently. The integrin-specific nanoparticles of the inventioncan target and block the binding of integrins exposed on smooth musclecells by arterial overstretch injury as well as deliver a variety oftherapeutic agents directly to cells that could inhibit inflammatory andrestenosis processes and provide for molecular imaging for new,prognostic data relating the extent and severity of balloon injury tosubsequent restenosis. The invention compositions avoid these problems.

Antibodies that are specific for α_(v)β₃ integrin have been described inU.S. Pat. No. 6,171,588. These antibodies have been used in targetedmagnetic resonance imaging (MRI) in a report by Sipkins, D. A., et al.,Nature Med. (1998) 4:623-626; in this case coupled to the surface ofliposomes via avidin linker proteins.

The use of antibodies directed to α_(v)β₃ as a targeting agent for MRIusing perfluorocarbon emulsions carrying chelated gadolinium has alsobeen described by Anderson, S. A., et al., Magn. Reson. Med. (2000)44:433-439, and in the above noted PCT publication WO 02/060524. Peptideligands that are targeted to integrins have also been used asantagonists and have been suggested as a therapeutic strategy forrheumatoid arthritis by Storgard, C. M., et al., J. Clin. Invest. (1999)103:47-53, who employed cyclic peptides containing the “RGD” typesequence known to interact with integrins.

Similar cyclic peptides were employed by Haubner, R., et al., J. Nucl.Med. (1999) 40:1061-1071 for tumor imaging by coupling the cyclicpeptides directly to radionuclides. In an additional paper, the use ofglycosylated forms of the cyclic peptides both for radiolabeling and PETis suggested by Haubner, R., et al., J. Nucl. Med. (2001) 42:326-336.

To applicants' knowledge, α_(v)β₃-specific moieties other thanantibodies have not been suggested for use as targeting agents indelivering image-aiding nanoparticulate emulsions or in deliveringemulsions containing bioactive agents to regions containing activatedendothelial cells such as sites of inflammation, tumors, atheroscleroticplaques, and restenoses.

DISCLOSURE OF THE INVENTION

The invention is directed to compositions and methods for imaging anddrug delivery wherein non-antibody, α_(v)β₃-specific moieties are usedas targeting agents to deliver nanoparticle emulsions to regionscontaining high levels of angiogenesis, such as tumors, regions ofinflammation, atherosclerotic regions, and restenoses. The use of theseagents in the context of imaging nanoparticle emulsions results inimproved image quality and the opportunity for targeted drug delivery.

Thus, in one aspect, the invention is directed to a method to deliver ananoparticulate emulsion to a target tissue, wherein said target tissueis characterized by high levels of α_(v)β₃ which method comprisesadministering to a subject comprising such tissue an emulsion ofnanoparticles wherein said nanoparticles are coupled to a ligandspecific for α_(v)β₃, with the proviso that said ligand is other than anantibody or fragment thereof.

In other aspects, the invention is directed to compositions useful inthe method of the invention, and to kits containing components of thecompositions that can be assembled to perform the invention methods. Thekits will typically provide emulsions that contain reactive groups thatcan bind to targeting agents provided separately, or that can bind toancillary substances useful for imaging or drug delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the particle size distribution of α_(v)β₃ targeted andnon-targeted nanoparticles.

FIG. 2 shows an enlarged section of T₁-weighted magnetic resonance imageof an implanted Vx-2 tumor.

FIG. 3 shows histological sections of Vx-2 tumor with H&E staining (lowpower magnification) and α_(v)β₃ staining (inset, high-powermagnification).

FIG. 4 is a graph showing enhancement in ROI from tumor (top) and muscle(bottom) in subjects receiving either targeted or non-targetednanoparticles.

FIG. 5A-5C show various magnification levels of histological sections ofinflammation in tumor slices.

FIG. 6 shows T₂-weighted and T₁-weighted MRI of tumors targeted withα_(v)β₃ targeted nanoparticles.

FIG. 7A shows a spin-echo image of aortic slices before and afteradministration of α_(v)β₃ labeled particles. FIG. 7B shows theenhancement of image in aorta of cholesterol treated subjects, untreatedsubjects, and in cholesterol treated subjects with non-targetedemulsions.

FIGS. 8A and 8B show the percent enhancement of MRI signal in aorta andmuscle using targeted and non-targeted particles.

FIG. 9 shows a 3-D angiogram of carotid arteries of domestic pigsfollowing angioplasty with α_(v)β₃-targeted paramagnetic nanoparticlesillustrating balloon overstretch injury pattern.

MODES OF CARRYING OUT THE INVENTION

The present invention offers an approach whereby superior imaging ofsites of activated endothelial cell concentrations can be obtained.Various emulsions which are useful in imaging can be employed. When usedalone, the nanoparticle-containing emulsions are useful as contrastagents for ultrasound imaging. For use in magnetic resonance imaging orin X-ray imaging, it may be desirable to employ a transition metal as acontrast agent; if the nanoparticles comprise fluorocarbons, however,the fluorocarbon itself is useful in obtaining an image. Radionuclidesare also useful both as diagnostic and therapeutic agents. In addition,reagents for optical imaging, such as fluorophores may also beassociated with the nanoparticles. In addition, or alternatively, thenanoparticles in the emulsion may contain one or more bioactive agents.

Any nanoparticulate emulsion may be used. For example, PCT publicationWO95/03829 describes oil emulsions where the drug is dispersed orsolubilized inside an oil droplet and the oil droplet is targeted to aspecific location by means of a ligand. U.S. Pat. No. 5,542,935describes site-specific drug delivery using gas-filled perfluorocarbonmicrospheres. The drug delivery is accomplished by permitting themicrospheres to home to the target and then effecting their rupture. Lowboiling perfluoro compounds are used to form the particles so that thegas bubbles can form.

However, it is preferred to employ emulsions wherein the nanoparticlesare based on high boiling perfluorocarbon liquids such as thosedescribed in U.S. Pat. No. 5,958,371 referenced above. The liquidemulsion contains nanoparticles comprised of relatively high boilingperfluorocarbons surrounded by a coating which is composed of a lipidand/or surfactant. The surrounding coating is able to couple directly toa targeting moiety or can entrap an intermediate component which iscovalently coupled to the targeting moiety, optionally through a linker,or may contain a non-specific coupling agent such as biotin.Alternatively, the coating may be cationic so that negatively chargedtargeting agents such as nucleic acids, in general or aptamers, inparticular, can be adsorbed to the surface.

In addition to the targeting α_(v)β₃ ligand, the nanoparticles maycontain associated with their surface an “ancillary agent” useful inimaging and/or therapy a radionuclide, a contrast agent for magneticresonance imaging (MRI) or for X-ray imaging, a fluorophore and/or abiologically active compound. The nanoparticles themselves can serve ascontrast agents for ultrasound imaging.

The preferred emulsion is a nanoparticulate system containing a highboiling perfluorocarbon as a core and an outer coating that is alipid/surfactant mixture which provides a vehicle for binding amultiplicity of copies of one or more desired components to thenanoparticle. The construction of the basic particles and the formationof emulsions containing them, regardless of the components bound to theouter surface is described in the above-cited patents to the presentapplicants, U.S. Pat. Nos. 5,690,907 and 5,780,010; and patents issuedon daughter U.S. Pat. Nos. 5,989,520 and 5,958,371 and incorporatedherein by reference.

The high boiling fluorochemical liquid is such that the boiling point ishigher than that of body temperature—i.e., 37° C. Thus, fluorochemicalliquids which have boiling points at least 30° C. are preferred, morepreferably 37° C., more preferably above 50° C., and most preferablyabove about 90° C. The “fluorochemical liquids” useful in the inventioninclude straight and branched chain and cyclic perfluorocarbonsincluding perfluorinated compounds which have other functional groups.“Perfluorinated compounds” includes compounds that are not pureperfluorocarbons but rather wherein other halo groups may be present.These include perfluorooctylbromide, and perfluorodichlorooctane, forexample.

Perfluorinated compounds as thus defined are preferred.

Useful perfluorocarbon emulsions are disclosed in U.S. Pat. Nos.4,927,623, 5,077,036, 5,114,703, 5,171,755, 5,304,325, 5,350,571,5,393,524, and 5,403,575, which are incorporated herein by reference,and include those in which the perfluorocarbon compound isperfluorodecalin, perfluorooctane, perfluorodichlorooctane,perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane,perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine,perfluortributylamine, perfluorodimethylcyclohexane,perfluorotrimethylcyclohexane, perfluorodicyclohexyl ether,perfluoro-n-butyltetrahydrofuran, and compounds that are structurallysimilar to these compounds and are partially or fully halogenated(including at least some fluorine substituents) or partially or fullyfluorinated including perfluoroalkylated ether, polyether or crownether.

The lipid/surfactants used to form an outer coating on the nanoparticles(that will contain the coupled ligand or entrap reagents for bindingdesired components to the surface) include natural or syntheticphospholipids, fatty acids, cholesterols, lysolipids, sphingomyelins,and the like, including lipid conjugated polyethylene glycol. Variouscommercial anionic, cationic, and nonionic surfactants can also beemployed, including Tweens, Spans, Tritons, and the like. Somesurfactants are themselves fluorinated, such as perfluorinated alkanoicacids such as perfluorohexanoic and perfluorooctanoic acids,perfluorinated alkyl sulfonamide, alkylene quaternary ammonium salts andthe like. In addition, perfluorinated alcohol phosphate esters can beemployed. Cationic lipids included in the outer layer may beadvantageous in entrapping ligands such as nucleic acids, in particularaptamers. Typical cationic lipids may include DOTMA,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP,1,2-dioleoyloxy-3-(trimethylammonio)propane; DOTB,1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol,1,2-diacyl-3-trimethylammonium-propane;1,2-diacyl-3-dimethylammonium-propane; 1,2-diacyl-sn-glycerol-3-ethylphosphocholine; and3β-[N′,N′-dimethylaminoethane)-carbamol]cholesterol-HCl.

In preferred embodiments, included in the lipid/surfactant coating arecomponents with reactive groups that can be used to couple the α_(v)β₃ligand and/or the ancillary substance useful for imaging or therapy. Aswill be described below, the lipid/surfactant components can be coupledto these reactive groups through functionalities contained in thelipid/surfactant component. For example, phosphatidylethanolamine may becoupled through its amino group directly to a desired moiety, or may becoupled to a linker such as a short peptide which may provide carboxyl,amino, or sulfhydryl groups as described below. Alternatively, standardlinking agents such a maleimides may be used. A variety of methods maybe used to associate the targeting ligand and the ancillary substancesto the nanoparticles; these strategies may include the use of spacergroups such as polyethyleneglycol or peptides, for example.

The lipid/surfactant coated nanoparticles are typically formed bymicrofluidizing a mixture of the fluorocarbon lipid which forms the coreand the lipid/surfactant mixture which forms the outer layer insuspension in aqueous medium to form an emulsion. In this procedure, thelipid/surfactants may already be coupled to additional ligands when theyare coated onto the nanoparticles, or may simply contain reactive groupsfor subsequent coupling. Alternatively, the components to be included inthe lipid/surfactant layer may simply be solubilized in the layer byvirtue of the solubility characteristics of the ancillary material.Sonication or other techniques may be required to obtain a suspension ofthe lipid/surfactant in the aqueous medium. Typically, at least one ofthe materials in the lipid/surfactant outer layer comprises a linker orfunctional group which is useful to bind the additional desiredcomponent or the component may already be coupled to the material at thetime the emulsion is prepared.

For coupling by covalently binding the targeting ligand or other organicmoiety (such as a chelating agent for a paramagnetic metal) to thecomponents of the outer layer, various types of bonds and linking agentsmay be employed. Typical methods for forming such coupling includeformation of amides with the use of carbodiamides, or formation ofsulfide linkages through the use of unsaturated components such asmaleimide. Other coupling agents include, for example, glutaraldehyde,propanedial or butanedial, 2-iminothiolane hydrochloride, bifunctionalN-hydroxysuccinimide esters such as disuccinimidyl suberate,disuccinimidyl tartrate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone,heterobifunctional reagents such asN-(5-azido-2-nitrobenzoyloxy)succinimide, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and succinimidyl4-(p-maleimidophenyl)butyrate, homobifunctional reagents such as1,5-difluoro-2,4-dinitrobenzene,4,4′-difluoro-3,3′-dinitrodiphenylsulfone,4,4′-diisothiocyano-2,2′-disulfonic acid stilbene,p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenylester), 4,4′-dithiobisphenylazide, erythritolbiscarbonate andbifunctional imidoesters such as dimethyl adipimidate hydrochloride,dimethyl suberimidate, dimethyl 3,3′-dithiobispropionimidatehydrochloride and the like. Linkage can also be accomplished byacylation, sulfonation, reductive amination, and the like. Amultiplicity of ways to couple, covalently, a desired ligand to one ormore components of the outer layer is well known in the art. The liganditself may be included in the surfactant layer if its properties aresuitable. For example, if the ligand contains a highly lipophilicportion, it may itself be embedded in the lipid/surfactant coating.Further, if the ligand is capable of direct adsorption to the coating,this too will effect its coupling. For example, nucleic acids, becauseof their negative charge, adsorb directly to cationic surfactants.

The ligand may bind directly to the nanoparticle, i.e., the ligand isassociated with the nanoparticle itself. Alternatively, indirect bindingsuch as that effected through biotin/avidin may be employed typicallyfor the α_(v)β₃-specific ligand. For example, in biotin/avidin mediatedtargeting, the α_(v)β₃ ligand is coupled not to the emulsion, but rathercoupled, in biotinylated form to the targeted tissue.

Ancillary agents that may be coupled to the nanoparticles throughentrapment in the coating layer include radionuclides. Radionuclides maybe either therapeutic or diagnostic; diagnostic imaging using suchnuclides is well known and by targeting radionuclides to undesiredtissue a therapeutic benefit may be realized as well. Typical diagnosticradionuclides include ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga,and therapeutic nuclides include ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir. The nuclide can beprovided to a preformed emulsion in a variety of ways. For example,⁹⁹Tc-pertechnate may be mixed with an excess of stannous chloride andincorporated into the preformed emulsion of nanoparticles. Stannousoxinate can be substituted for stannous chloride. In addition,commercially available kits, such as the HM-PAO (exametazine) kitmarketed as Ceretek® by Nycomed Amersham can be used. Means to attachvarious radioligands to the nanoparticles of the invention areunderstood in the art.

Chelating agents containing paramagnetic metals for use in magneticresonance imaging can also be employed as ancillary agents. Typically, achelating agent containing a paramagnetic metal is associated with thelipids/surfactants of the coating on the nanoparticles and incorporatedinto the initial mixture which is sonicated. The chelating agent can becoupled directly to one or more of components of the coating layer.Suitable chelating agents include a variety of multi-dentate compoundsincluding EDTA, DPTA, DOTA, and the like. These chelating agents can becoupled directly to functional groups contained in, for example,phosphatidyl ethanolamine, bis-oleate, and the like, or through linkinggroups.

The paramagnetic metals useful in the MRI contrast agents of theinvention include rare earth metals, typically, manganese, ytterbium,gadolinium, europium, and the like. Iron ions may also be used.

Other ancillary agents include fluorophores such as fluorescein, dansyl,quantum dots, and the like.

Included in the surface of the nanoparticle, in some embodiments of theinvention, are biologically active agents. These biologically activeagents can be of a wide variety, including proteins, nucleic acids,pharmaceuticals, and the like. Thus, included among suitablepharmaceuticals are antineoplastic agents, hormones, analgesics,anesthetics, neuromuscular blockers, antimicrobials or antiparasiticagents, antiviral agents, interferons, antidiabetics, antihistamines,antitussives, anticoagulants, and the like.

In all of the foregoing cases, whether the associated moiety is atargeting ligand for α_(v)β₃ or is an ancillary agent, the definedmoiety may be non-covalently associated with the lipid/surfactant layer,may be directly coupled to the components of the lipid/surfactant layer,or may be coupled to said components through spacer moieties.

Targeting Ligands

The emulsions of the present invention employ targeting agents that areligands specific for the α_(v)β₃ integrin other than an antibody orfragment thereof. In one embodiment, the ligand is a non-peptide organicmolecule, such as those described in U.S. Pat. Nos. 6,130,231;6,153,628; 6,322,770; and PCT publication WO 01/97848 referenced above,and incorporated herein by reference. “Non-peptide” moieties in generalare those other than compounds which are simply polymers of amino acids,either gene encoded or non-gene encoded. Thus, “non-peptide ligands” aremoieties which are commonly referred to as “small molecules” lacking inpolymeric character and characterized by the requirement for a corestructure other than a polymer of amino acids. The non-peptide ligandsuseful in the invention may be coupled to peptides or may includepeptides coupled to portions of the ligand which are responsible foraffinity to the α_(v)β₃ moiety, but it is the non-peptide regions ofthis ligand which account for its binding ability.

One group of α_(v)β₃-specific ligands that is particularly useful in themethods and compositions of the invention are of the formula (I):

including stereoisomeric forms thereof, or mixtures of stereoisomericforms thereof, or pharmaceutically acceptable salt or prodrug formsthereof, wherein:

Hc comprises guanidyl or comprises a heterocyclic ring containing N;

L¹ is a linker;

G is N or CR^(B);

R^(A) is a non-interfering substituent other than H;

each R^(B) is independently H or a non-interfering substituent; and

M comprises an optionally substituted carboxylic, sulfonylic, orphosphoric acid group or an ester or amide thereof or is a 4- or5-membered ring;

wherein each of ring A and ring B may optionally further be substitutedwith non-interfering substituents.

When appropriate, the compounds may be in the form of salts.

When the compounds of Formula (I) contain one or more chiral centers,the invention includes optically pure forms as well as mixtures ofstereoisomers or enantiomers.

In the compounds of formula (I), the carboxylic, sulfonylic orphosphoric acid groups or esters or amides thereof included in M may bepositioned in either orientation with respect to the molecule—i.e., asulfonamide may be SO₂N— or —NSO₂—; in addition, multiple carboxylic,sulfonylic or phosphoric acid groups, esters or amides thereof may beincluded in tandem. These residues may further be substituted, and maybe coupled to the components of the nanoparticles through variouslinking groups, including those which contain PEG and those that containpeptide linkages.

Preferred embodiments of M are selected from the group consisting of—COR^(B), —SO₃H, —PO₃H, —CONHNHSO₂CF₃, —CONHSO₂R^(B), —CONHSO₂NHR^(B),—NHCOCF₃, —NHCONHSO₂R^(B), —NHSO₂R^(B), —OPO₃H₂, —OSO₃H, —PO₃H₂,—SO₂NHCOR^(B), —SO₂NHCO₂R^(B),

A “non-interfering substituent” is a substituent which does not destroythe ability of the compounds of formula (I) to bind to α_(v)β₃. Thesubstituent may alter the strength of binding, but the binding muststill be detectable using standard methods, such as detection of labelbound to a solid support wherein the solid support is coupled toα_(v)β₃. The essential features of the compounds of formula (I) are setforth in the formula, and clearly a variety of substituents may furtherbe included without even substantially altering the ability of thecompound thus to bind. The skilled artisan can readily assess, for anyparticular embodiment of R^(B) whether the binding characteristics toα_(v)β₃ are sufficiently satisfactory to warrant the incorporation ofthe R^(B) embodiment tested. Thus, for any arbitrarily chosenembodiment, it is a straightforward matter to determine whether thesubstituent interferes or does not interfere.

Thus, the essential features of the molecule are tightly defined. Thepositions which are occupied by “noninterfering substituents” can besubstituted by conventional inorganic or organic moieties as isunderstood in the art. It is irrelevant to the present invention to testthe outer limits of such substitutions. The essential features of thecompounds are those set forth with particularity herein.

In addition, L¹ is described herein as a linker. The nature of such alinker is less important that the distance it imparts between theportions of the molecule. Typical linkers include alkylene, i.e.(CH₂)_(n); alkenylene—i.e., an alkylene moiety which contains a doublebond, including a double bond at one terminus. Other suitable linkersinclude, for example, substituted alkylenes or alkenylenes, carbonylmoieties, and the like.

“Hydrocarbyl residue” refers to a residue which contains only carbon andhydrogen. The residue may be aliphatic or aromatic, straight-chain,cyclic, branched, saturated or unsaturated. The hydrocarbyl residue,when so stated however, may contain heteroatoms over and above, orsubstituted for, the carbon and hydrogen members of the substituentresidue. Thus, when specifically noted as containing such heteroatoms,the hydrocarbyl residue may also contain carbonyl groups, amino groups,hydroxyl groups and the like, or contain heteroatoms within the“backbone” of the hydrocarbyl residue.

“Alkyl,” “alkenyl” and “alkynyl” include straight- and branched-chainand cyclic monovalent substituents. Examples include methyl, ethyl,isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and thelike. Typically, the alkyl, alkenyl and alkynyl substituents contain1-10C (alkyl) or 2-10C (alkenyl or alkynyl). Preferably they contain1-6C (alkyl) or 2-6C (alkenyl or alkynyl). Such moieties containingheteroatoms are similarly defined but may contain 1-2 O, S, P₁Si or Nheteroatoms or combinations thereof within the backbone residue.

“Acyl” encompasses the definitions of alkyl, alkenyl, alkynyl and therelated hetero-forms which are coupled to an additional residue througha carbonyl group.

“Aromatic” or “aryl” moiety refers to a monocyclic or fused bicyclicmoiety such as phenyl or naphthyl; “heteroaromatic” also refers tomonocyclic or fused bicyclic ring systems containing one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits inclusion of 5-membered rings as well as 6-membered rings. Thus,typical aromatic systems include pyridyl, pyrimidyl, indolyl,benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl,benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyland the like. Any monocyclic or fused ring bicyclic system which has thecharacteristics of aromaticity in terms of electron distributionthroughout the ring system is included in this definition. Typically,the ring systems contain 5-12 ring member atoms.

The “non-interfering substituents” are typically halo, OH, SH, NH₂, NO₂,or other inorganic substituents or are hydrocarbyl residues (1-20C)containing 0-6 heteroatoms selected from O, S, P, Si, and N. Preferablythe heteroatoms are O, S and/or N. For example, the hydrocarbyl residuemay be alkyl, alkenyl, alkynyl, aryl, arylalkyl, which substituents maycontain the above mentioned heteroatoms and/or may themselves besubstituted with 1-6 substituents. The substituents on aryl moieties oron suitable heteroatoms include alkyl, alkenyl, alkynyl, additionalaryl, or arylalkyl, arylalkenyl, and arylalkynyl. Substituents which mayoccur on non-cyclic carbon chains, including appropriate heteroatoms,include substituted forms of these moieties and/or heteroatom-containingforms thereof as well as halo, OR, NR₂, SR, SOR, SO₂R, OCOR, NRCOR,NRCONR₂, NRCOOR, OCONR₂, RCO, COOR, SO₃R, CONR₂, SO₂NR₂, NRSO₂NR₂, CN,CF₃, R₃Si, and NO₂ where each R is independently alkyl, alkenyl, aryl,etc., or heteroforms thereof. Two substituents may form a ring or ═O.

Two R^(B) on adjacent positions or on the same C or N can be joined toform a fused, optionally substituted aromatic or nonaromatic saturatedor unsaturated ring which contains 3-8 members or two R^(B) may be ═O oran oxime, oxime ether, oxime ester or ketal thereof.

In one set of embodiments, in the compounds of formula (I), Hc is anoptionally substituted 5 or 6 membered ring containing one or twonitrogens. Preferred substituents include amines.

One set of embodiments of L¹ includes alkylene chains of 1-4 memberatoms of which one or two non-adjacent members may be heteroatoms whichare N, S or O, preferably N. Preferred embodiments for G include N andCH.

Preferred embodiments of R include H, alkyl (1-10C), alkenyl (2-10C),acyl (1-10C), arylalkyl or arylacyl wherein alkyl and acyl are definedas above and aryl contains 5-12 ring members including, optionally,heteroatoms selected from N, O and S. When R^(B) is substituted onto acarbon, R^(B) may be COOR (where R is H or alkyl (1-10C), or CONR₂wherein R is as previously defined, OOCR or NROCR where R is aspreviously defined, halo, CF₃, and the like.

One group of α_(v)β₃-specific ligands that are embodiments of formula(I) useful in the invention are compounds of the formula (II):

including stereoisomeric forms thereof, or mixtures of stereoisomericforms thereof, or pharmaceutically acceptable salt or prodrug formsthereof,

-   wherein R^(1e) is selected from:

-   -   A^(e) is —CH₂— or —N(R^(10e))—;    -   A^(1e) and B^(e) are independently —CH₂— or —N(R^(10e))—;    -   D^(e) is —N(R^(10e))— or —S—;    -   E^(e)-F^(e) is —C(R^(2e))═C(R^(3e))— or —C(R^(2e))₂C(R^(3e))₂—;    -   J^(e) is —C(R^(2e))— or —N—;    -   K^(e), L^(e) and M^(e) are independently —C(R^(2e))— or        —C(R^(3e))—;    -   R^(2e) and R^(3e) are independently selected from:    -   H, C₁-C₄ alkoxy, NR^(11e)R^(12e), halogen, NO₂, CN, CF₃, C₁-C₆        alkyl, C₃-C₆ alkenyl, C₃-C₇ cycloalkyl, C₃-C₇ cycloalkyl(C₁-C₄        alkyl), aryl(C₁-C₆ alkyl)-, (C₁-C₆ alkyl)carbonyl, (C₁-C₆        alkoxy)carbonyl, arylcarbonyl, and aryl substituted with 0-4        R^(7e),    -   alternatively, when R^(2e) and R^(3e) are substituents on        adjacent atoms, they can be taken together with the carbon atoms        to which they are attached to form a 5-7 membered carbocyclic or        5-7 membered heterocyclic aromatic or nonaromatic ring system,        said carbocyclic or heterocyclic ring being substituted with 0-2        groups selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, halo, cyano,        amino, CF₃ and NO₂;    -   R^(2ae) is selected from:    -   H, C₁-C₁₀ alkyl, C₂-C₆ alkenyl, C₃-C₁₁ cycloalkyl, C₃-C₇        cycloalkyl(C₁-C₄ alkyl), aryl, aryl(C₁-C₄ alkyl)-, (C₂-C₇        alkyl)carbonyl, arylcarbonyl, (C₂-C₁₀ alkoxy)carbonyl, C₃-C₇        cycloalkoxycarbonyl, C₇-C₁₁ bicycloalkoxycarbonyl,        aryloxycarbonyl, aryl(C₁-C₁₀ alkoxy)carbonyl, C₁-C₆        alkylcarbonyloxy(C₁-C₄ alkoxy)carbonyl, arylcarbonyloxy(C₁-C₄        alkoxy)carbonyl, and C₃-C₇ cycloalkylcarbonyloxy(C₁-C₄        alkoxy)carbonyl;    -   R^(7e) is selected from:    -   H, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, aryl, aryl(C₁-C₄ alkyl)-,        (C₁-C₄ alkyl)carbonyl, CO₂R^(18ae), SO₂R^(11e),        SO₂NR^(10e)R^(11e), OR^(10e), and N(R^(11e))R^(12e);

-   wherein U^(e) is selected from:    -   —(CH₂)_(n) ^(e)—, —(CH₂)_(n) ^(e)O(CH₂)_(m) ^(e)—,        —(CH₂)neN(R¹²)(CH₂)_(m) ^(e)—, —NH(CH₂)_(n) ^(e)—, —(CH₂)_(n)        ^(e)C(═O)(CH₂)_(m) ^(e)—, —(CH₂)_(n) ^(e)S(O)_(p) ^(e)(CH₂)_(m)        ^(e)—, —(CH₂)_(n) ^(e)NHNH(CH₂)_(m) ^(e)—, —N(R^(10e))C(═O)—,        —NHC(═O)(CH₂)_(n) ^(e)—, —C(═O)N(R^(10e))—, and        —N(R^(10e))S(O)_(p) ^(e)—;

-   wherein G^(e) is N or CR^(19e);

-   wherein W^(e) is —C(═O)—N(R^(10e))—(C₁-C₃ alkylene)-, in which the    alkylene group is substituted by R^(8e) and by R^(9e):    -   R^(8e) and R^(9e) are independently selected from:    -   H, CO₂R^(18be), C(═O)R^(18be), CONR¹⁷R^(18be), C₁-C₁₀ alkyl        substituted with 0-1 R^(6e), C₂-C₁₀ alkenyl substituted with 0-1        R^(6e), C₂-C₁₀ alkynyl substituted with 0-1 R^(6e), C₃-C₈        cycloalkyl substituted with 0-1 R^(6e), C₅-C₆ cycloalkenyl        substituted with 0-1 R^(6e), (C₁-C₁₀ alkyl)carbonyl, C₃-C₁₀        cycloalkyl(C₁-C₄ alkyl)-, phenyl substituted with 0-3 R^(6e),        naphthyl substituted with 0-3 R^(6e),    -   a 5-10 membered heterocyclic ring containing 1-3 N, O, or S        heteroatoms, wherein said heterocyclic ring may be saturated,        partially saturated, or fully unsaturated, said heterocyclic        ring being substituted with 0-2 R^(7e),    -   C₁-C₁₀ alkoxy substituted with 0-2 R^(7e), hydroxy, nitro,        —N(R^(10e))R^(11e), —N(R^(16e))R^(17e), aryl(C₀-C₆        alkyl)carbonyl, aryl(C₃-C₆ alkyl), heteroaryl(C₁-C₆ alkyl),        CONR^(18ae)R^(20e), SO₂R^(18ae), and SO₂NR^(18ae)R^(20e),    -   providing that any of the above alkyl, cycloalkyl, aryl or        heteroaryl groups may be unsubstituted or substituted        independently with 1-2 R^(7e);    -   R^(6e) is selected from:    -   H, C₁-C₁₀ alkyl, hydroxy, C₁-C₁₀ alkoxy, nitro, C₁-C₁₀        alkylcarbonyl, —N(R^(11e))R^(12e), cyano, halo, CF₃, CHO,        CO₂R^(18be), C(═O)R^(18be), CONR^(17e)R^(18be), OC(═O)R^(10e),        OR^(10e), OC(═O)NR^(10e)R^(11e), NR^(10e)C(═O)R^(10e),        NR^(10e)C(═O)OR^(21e), NR^(10e)C(═O)NR^(10e)R^(11e),        NR^(10e)SO₂NR^(10e)R^(11e), NR^(10e)SO₂R^(21e), S(O)_(p)R^(11e),        SO₂NR^(10e)R^(11e),    -   aryl substituted with 0-3 groups selected from halogen, C₁-C₆        alkoxy, C₁-C₆ alkyl, CF₃, S(O)_(m) ^(e)Me, and —NMe₂,    -   aryl(C₁-C₄ alkyl)-, said aryl being substituted with 0-3 groups        selected from halogen, C₁-C₆ alkoxy, C₁-C₆ alkyl, CF₃, S(O)_(p)        ^(e)Me, and —NMe₂, and    -   a 5-10 membered heterocyclic ring containing 1-3 N, O, or S        heteroatoms, wherein said heterocyclic ring may be saturated,        partially saturated, or fully unsaturated, said heterocyclic        ring being substituted with 0-2 R^(7e);    -   R^(10e) is selected from:    -   H, CF₃, C₃-C₆ alkenyl, C₃-C₁₁ cycloalkyl, aryl, (C₃-C₁₁        cycloalkyl)methyl, aryl(C₁-C₄ alkyl), and C₁-C₁₀ alkyl        substituted with 0-2 R^(6e);    -   R^(11e) is selected from:    -   H, hydroxy, C₁-C₈ alkyl, C₃-C₆ alkenyl, C₃-C₁₁ cycloalkyl,        (C₃-C₁₁ cycloalkyl)methyl, C₁-C₆ alkoxy, benzyloxy, aryl,        heteroaryl, heteroaryl(C₁-C₄ alkyl)-, aryl(C₁-C₄ alkyl),        adamantylmethyl, and C₁-C₁₀ alkyl substituted with 0-2 R^(4e);    -   R^(4e) is selected from:    -   H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl, C₃-C₇ cycloalkyl(C₁-C₄        alkyl)-, (C₁-C₁₀ alkyl)carbonyl, aryl, heteroaryl, aryl(C₁-C₆        alkyl)-, and heteroaryl(C₁-C₆ alkyl)-, wherein said aryl or        heteroaryl groups are substituted with 0-2 substituents        independently selected from the group consisting of C₁-C₄ alkyl,        C₁-C₄ alkoxy, F, Cl, Br, CF₃, and NO₂,    -   alternatively, when R^(10e) and R^(11e) are both substituents on        the same nitrogen atom (as in —NR^(10e)R^(11e)) they may be        taken together with the nitrogen atom to which they are attached        to form a heterocycle selected from: 3-azabicyclononyl,        1,2,3,4-tetrahydro-1-quinolinyl,        1,2,3,4-tetrahydro-2-isoquinolinyl, 1-piperidinyl,        1-morpholinyl, 1-pyrrolidinyl, thiamorpholinyl, thiazolidinyl,        and 1-piperazinyl;    -   said heterocycle being substituted with 0-3 groups selected        from: C₁-C₆ alkyl, aryl, heteroaryl, aryl(C₁-C₄ alkyl)-, (C₁-C₆        alkyl)carbonyl, (C₃-C₇ cycloalkyl)carbonyl, (C₁-C₆        alkoxy)carbonyl, aryl(C₁-C₄ alkoxy)carbonyl, C₁-C₆        alkylsulfonyl, and arylsulfonyl;    -   R^(12e) is selected from:    -   H, C₁-C₆ alkyl, triphenylmethyl, methoxymethyl,        methoxyphenyldiphenylmethyl, trimethylsilylethoxymethyl, (C₁-C₆        alkyl)carbonyl, (C₁-C₆ alkoxy)carbonyl, (C₁-C₆        alkyl)aminocarbonyl, C₃-C₆ alkenyl, C₃-C₇ cycloalkyl, C₃-C₇        cycloalkyl(C₁-C₄ alkyl)-, aryl, heteroaryl(C₁-C₆ alkyl)carbonyl,        heteroarylcarbonyl, aryl(C₁-C₆ alkyl)-, (C₁-C₆ alkyl)carbonyl,        arylcarbonyl, C₁-C₆ alkylsulfonyl, arylsulfonyl, aryl(C₁-C₆        alkyl)sulfonyl, heteroarylsulfonyl, heteroaryl(C₁-C₆        alkyl)sulfonyl, aryloxycarbonyl, and aryl(C₁-C₆ alkoxy)carbonyl,        wherein said aryl groups are substituted with 0-2 substituents        selected from the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy,        halo, CF₃, and nitro;    -   R^(16e) is selected from:    -   —C(═O—)O)R^(18ae), —C(═O)R^(18be), —C(═O)N(R^(18be))₂,        —C(═O)NHSO₂R^(18ae), —C(═O)NHC(═O)R^(18be),        —C(═O)NHC(═O)OR^(18ae), —C(═O)NHSO₂NHR^(18be), —SO₂R^(18ae),        —SO₂N(R^(18be))₂, and —SO₂NHC(═O)OR^(18be);    -   R^(17e) is selected from:    -   H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl, C₃-C₇ cycloalkyl(C₁-C₄        alkyl)-, aryl, aryl(C₁-C₆ alkyl)-, and heteroaryl(C₁-C₆ alkyl);

-   wherein R^(18ae) is selected from:    -   C₁-C₈ alkyl optionally substituted with a bond to L_(n), C₃-C₁₁        cycloalkyl optionally substituted with a bond to L_(n),        aryl(C₁-C₆ alkyl)- optionally substituted with a bond to L_(n),        heteroaryl(C₁-C₆ alkyl)- optionally substituted with a bond to        L_(n), (C₁-C₆ alkyl)heteroaryl optionally substituted with a        bond to L_(n), biaryl(C₁-C₆ alkyl) optionally substituted with a        bond to L_(n), heteroaryl optionally substituted with a bond to        L_(n), phenyl substituted with 3-4 R^(19e) and optionally        substituted with a bond to L_(n), naphthyl substituted with 0-4        R^(19e) and optionally substituted with a bond to L_(n), and a        bond to L_(n), wherein said aryl or heteroaryl groups are        optionally substituted with 0-4 R^(19e);    -   R^(18be) is H or R^(18ae);

-   wherein R^(19e) is selected from:    -   H, halogen, CF₃, CO₂H, CN, NO₂, —NR^(11e)R^(12e), OCF₃, C₁-C₈        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₁ cycloalkyl, C₃-C₇        cycloalkyl(C₁-C₄ alkyl)-, aryl(C₁-C₆ alkyl)-, C₁-C₆ alkoxy,        C₁-C₄ alkoxycarbonyl, aryl, aryl-O—, aryl-SO₂—, heteroaryl, and        heteroaryl-SO₂—, wherein said aryl and heteroaryl groups are        substituted with 0-4 groups selected from hydrogen, halogen,        CF₃, C₁-C₃ alkyl, and C₁-C₃ alkoxy;    -   R^(20e) is selected from:    -   hydroxy, C₁-C₁₀ alkyloxy, C₃-C₁₁ cycloalkyloxy, aryloxy,        aryl(C₁-C₄ alkyl)oxy, C₂-C₁₀ alkylcarbonyloxy(C₁-C₂ alkyl)oxy-,        C₂-C₁₀ alkoxycarbonyloxy(C₁-C₂ alkyl)oxy-, C₂-C₁₀        alkoxycarbonyl(C₁-C₂ alkyl)oxy-, C₃-C₁₀        cycloalkylcarbonyloxy(C₁-C₂ alkyl)oxy-, C₃-C₁₀        cycloalkoxycarbonyloxy(C₁-C₂ alkyl)oxy-, C₃-C₁₀        cycloalkoxycarbonyl(C₁-C₂ alkyl)oxy-, aryloxycarbonyl(C₁-C₂        alkyl)oxy-, aryloxycarbonyloxy(C₁-C₂ alkyl)oxy-,        arylcarbonyloxy(C₁-C₂ alkyl)oxy-, C₁-C₅ alkoxy(C₁-C₅        alkyl)carbonyloxy(C₁-C₂ alkyl)oxy, (5-(C₁-C₅        alkyl)-1,3-dioxa-cyclopenten-2-one-yl)methyloxy,        (5-aryl-1,3-dioxa-cyclopenten-2-one-yl)methyloxy, and        (R^(10e))(R^(11e))N—(C₁-C₁₀ alkoxy)-;    -   R^(21e) is selected from:    -   C₁-C₈ alkyl, C₂-C₆ alkenyl, C₃-C₁₁ cycloalkyl, (C₃-C₁₁        cycloalkyl)methyl, aryl, aryl(C₁-C₄ alkyl)-, and C₁-C₁₀ alkyl        substituted with 0-2 R^(7e);    -   R^(22e) is selected from: —C(═O)—R^(18be), —C(═O)N(R^(18be))₂,        —C(═O)NHSO₂R^(18ae), —C(═O)NHC(═O)R^(18be),        —C(═O)NHC(═O)OR^(18ae), and —C(═O)NHSO₂NHR^(18be);

-   wherein Y^(e) is selected from:    -   —COR^(20e), —SO₃H, —PO₃H, —CONHNHSO₂CF₃, —CONHSO₂R^(18ae),        —CONHSO₂NHR^(18be), —NHCOCF₃, —NHCONHSO₂R^(18ae),        —NHSO₂R^(18ae), —OPO₃H₂, —OSO₃H, —PO₃H₂, —SO₂NHCOR^(18ae),        —SO₂NHCO₂R^(18ae),

The ligand set forth above may be coupled through a linker to thematerials contained in the lipid/surfactant coating of the particles. Inone embodiment, the linkers are of the formula:

((W)_(h)—(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)—(W)_(h′))_(x′);

W is independently selected at each occurrence from the group: O, S, NH,NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)NR⁸, C(═O), C(═O)O, OC(═O), NHC(═S)NH,NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂O)₂₀₋₂₀₀,(OCH₂CH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂CH₂O)₂₀₋₂₀₀, and (aa)_(t);

aa is independently at each occurrence an amino acid;

Z is selected from the group: aryl substituted with 0-3 R¹⁰, C₃₋₁₀cycloalkyl substituted with 0-3 R¹⁰, and a 5-10 membered heterocyclicring system containing 1-4 heteroatoms independently selected from N, S,and O and substituted with 0-3 R¹⁰;

R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at eachoccurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkylsubstituted with 0-3 R¹⁰, aryl substituted with 0-3 R¹⁰, benzylsubstituted with 0-3 R¹⁰, and C₁-C₅ alkoxy substituted with 0-3 R¹⁰,NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to anadditional component;

R¹⁰ is independently selected at each occurrence from the group: a bondto S_(f), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H, PO₃H,—OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹¹, C₁₋₅ alkyl substitutedwith 0-1 R¹², C₁₋₅ alkoxy substituted with 0-1 R¹², and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-3 R¹¹;

R¹¹ is independently selected at each occurrence from the group: H,alkyl substituted with 0-1 R¹², aryl substituted with 0-1 R¹², a 5-10membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-1 R¹²,C₃₋₁₀ cycloalkyl substituted with 0-1 R¹², polyalkylene glycolsubstituted with 0-1 R¹², carbohydrate substituted with 0-1 R¹²,cyclodextrin substituted with 0-1 R¹², amino acid substituted with 0-1R¹², polycarboxyalkyl substituted with 0-1 R¹², polyazaalkyl substitutedwith 0-1 R¹², peptide substituted with 0-1 R¹², wherein the peptide iscomprised of 2-10 amino acids, 3,6-O-disulfo-B-D-galactopyranosyl,bis(phosphonomethyl)glycine, and a bond to an additional component;

R¹² is a bond to an additional component;

k is selected from 0, 1, and 2;

h is selected from 0, 1, and 2;

h′ is selected from 0, 1, and 2;

g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

x is selected from 0, 1, 2, 3, 4, and 5;

x′ is selected from 0, 1, 2, 3, 4, and 5;

In some embodiments, the additional substituent contained on thenanoparticles includes a chelator for a radionuclide or a metal forX-ray or magnetic resonance imaging. Such chelators include those of theformulas:

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected from:NR¹³, NR¹³R¹⁴, S, SH, S(Pg), O, OH, PR¹³, PR¹³R¹⁴, P(O)R¹⁵R¹⁶, and abond to the remainder of the complex;

E is a bond, CH, or a spacer group independently selected at eachoccurrence from the group: C₁-C₁₀ alkylene substituted with 0-3 R¹⁷,arylene substituted with 0-3 R¹⁷, C₃₋₁₀ cycloalkylene substituted with0-3 R¹⁷, heterocyclo-C₁₋₁₀ alkylene substituted with 0-3 R¹⁷, whereinthe heterocyclo group is a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O,C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with 0-3 R¹⁷, and a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-3 R¹⁷;

R¹³ and R¹⁴ are each independently selected from the group: a bond toL_(n′), hydrogen, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, arylsubstituted with 0-3 R¹⁷, C₁₋₁₀ cycloalkyl substituted with 0-3 R¹⁷,heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, wherein theheterocyclo group is a 5-10 membered heterocyclic ring system containing1-4 heteroatoms independently selected from N, S, and O, C₆₋₁₀aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀aryl-substituted with 0-3 R¹⁷, a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-3 R¹⁷, and an electron, provided that when one of R¹³or R¹⁴ is an electron, then the other is also an electron;

alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰)(R²¹);

R¹⁵ and R¹⁶ are each independently selected from the group: a bond toL_(n′), —OH, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, C₁-C₁₀ alkylsubstituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, C₃₋₁₀cycloalkyl substituted with 0-3 R¹⁷, heterocyclo-C₁₋₁₀ alkyl substitutedwith 0-3 R¹⁷, wherein the heterocyclo group is a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl- substituted with 0-3 R¹⁷, and a 5-10membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-3 R¹⁷;

R¹⁷ is independently selected at each occurrence from the group: a bondto L_(n′), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸,—C(═O)N(R¹⁸)₂, —CHO, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸,—OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R⁸, —NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂,—NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H, —SO₂R^(18a), —SR¹⁸,—S(═O)R^(18a), —SO₂N(R¹⁸)₂, —N(R¹⁸)₂, —NHC(═S)NHR¹⁸, ═NOR¹⁸, NO₂,—C(═O)NHOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁-C₅ alkyl, C₂-C₄ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylmethyl,C₂-C₆ alkoxyalkyl, aryl substituted with 0-2 R¹⁸, and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O;

R¹⁸, R^(18a), and R¹⁹ are independently selected at each occurrence fromthe group: a bond to L_(n′), H, C₁-C₆ alkyl, phenyl, benzyl, C₁-C₆alkoxy, halide, nitro, cyano, and trifluoromethyl;

Pg is a thiol protecting group;

R²⁰ and R²¹ are independently selected from the group: H, C₁-C₁₀ alkyl,—CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, C₂-C₁₀ 1-alkene substituted with0-3 R²³, C₂-C₁₀ 1-alkyne substituted with 0-3 R²³, aryl substituted with0-3 R²³, unsaturated 5-10 membered heterocyclic ring system containing1-4 heteroatoms independently selected from N, S, and O and substitutedwith 0-3 R²³, and unsaturated C₃₋₁₀ carbocycle substituted with 0-3 R²³;

alternatively, R²⁰ and R²¹, taken together with the divalent carbonradical to which they are attached form:

R²² and R²³ are independently selected from the group: H, R²⁴, C₁-C₁₀alkyl substituted with 0-3 R²⁴, C₂-C₁₀ alkenyl substituted with 0-3 R²⁴,C₂-C₁₀ alkynyl substituted with 0-3 R²⁴, aryl substituted with 0-3 R²⁴,a 5-10 membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-3 R²⁴,and C₃₋₁₀ carbocycle substituted with 0-3 R²⁴;

alternatively, R²², R²³ taken together form a fused aromatic or a 5-10membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O;

a and b indicate the positions of optional double bonds and n is 0 or 1;

R²⁴ is independently selected at each occurrence from the group: ═O, F,Cl, Br, I, —CF₃, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, —N(R²⁵)₃ ⁺,—CH₂OR²⁵, —OC(═O)R²⁵, —OC(═O)OR^(25a), —OR²⁵, —OC(═O)N(R²⁵)₂,—NR²⁶C(═O)R²⁵, —NR²⁶C(═O)OR^(25a), —NR²⁶C(═O)N(R²⁵)₂, —NR²⁶SO₂N(R²⁵)₂,—NR²⁶SO₂R^(25a), —SO₃H, —SO₂R^(25a), —SR²⁵, —S(═O)R^(25a), —SO₂N(R²⁵)₂,—N(R²⁵)₂, ═NOR²⁵, —C(═O)NHOR²⁵, —OCH₂CO₂H, and 2-(1-morpholino)ethoxy;and,

R²⁵, R^(25a), and R²⁶ are each independently selected at each occurrencefrom the group: hydrogen and C₁-C₆ alkyl;

and a pharmaceutically acceptable salt thereof.

In embodiment of the invention the α_(v)β₃ targeting moiety may be:

-   3-[7-[(imidazolin-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(3,5-dimethylisoxazol-4-ylsulfonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butylsulfonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butyloxycarbonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(phenylsulfonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butylsulfonyl)aminopropionic    acid,-   3-[7-[(2-aminothiazol-4-yl)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(3,5-dimethylisoxazol-4-ylsulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((4-biphenyl)sulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(1-naphthylsulfonylamino)propionic    acid,-   3-[7-[(benzimidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(4-methylimidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(4,5-dimethylimidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(4,5,6,7-tetrahydrobenzimidazol-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(pyridin-2-ylamino)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-(2-aminopyridin-6-yl)-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(7-azabenzimidazol-2-yl)methyl]-1-methyl-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(benzimidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]propionic    acid,-   3-[7-[(pyridin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(phenylsulfonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butylsulfonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butyloxycarbonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(phenylsulfonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(n-butylsulfonyl)aminopropionic    acid,-   3-[7-[(2-aminothiazol-4-yl)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(phenylsulfonylamino)propionic    acid,-   3-[7-[(2-aminothiazol-4-yl)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazolin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(tetrahydropyrimid-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfon-ylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(benzyloxycarbonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-(phenylsulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,6,dichlorophenyl)sulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(imidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((4-biphenyl)sulfonylamino)propionic    acid,-   3-[7-[(benzimidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(4-methylimidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(4,5-dimethylimidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(4,5,6,7-tetrahydrobenzimidazol-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-[(pyridin-2-ylamino)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid,-   3-[7-(2-aminopyridin-6-yl)-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid, or-   3-[7-[(7-azabenzimidazol-2-yl)methyl]-1-(2-phenylethyl)-6,8-difluoroquinoline-4-one-3-ylcarbonylamino]-2-((2,4,6-trimethylphenyl)sulfonylamino)propionic    acid.

Preparation Methods

In a typical procedure for preparing the emulsions of the invention, thefluorochemical liquid and the components of the lipid/surfactant coatingare fluidized in aqueous medium to form an emulsion. The functionalcomponents of the surface layer may be included in the originalemulsion, or may later be covalently coupled to the surface layersubsequent to the formation of the nanoparticle emulsion. In oneparticular instance, for example, where a nucleic acid targeting agentor drug is to be included, the coating may employ a cationic surfactantand the nucleic acid adsorbed to the surface after the particle isformed.

When appropriately prepared, the nanoparticles that comprise ancillaryagents contain a multiplicity of functional such agents at their outersurface, the nanoparticles typically contain hundreds or thousands ofmolecules of the biologically active agent, targeting ligand,radionuclide and/or MRI contrast agent. For MRI contrast agents, thenumber of copies of a component to be coupled to the nanoparticle istypically in excess of 5,000 copies per particle, more preferably 10,000copies per particle, still more preferably 30,000, and still morepreferably 50,000-100,000 or more copies per particle. The number oftargeting agents per particle is typically less, of the order of severalhundred while the concentration of fluorophores, radionuclides, andbiologically active agents is also variable.

The nanoparticles need not contain an ancillary agent. In general, thetargeted particles, directly coupled to a α_(v)β₃-specific ligand, areuseful themselves as ultrasound contrast agents. Further, because theparticles have a fluorocarbon core, ¹⁹F magnetic resonance imaging canbe used to track the location of the particles concomitantly with theiradditional functions described above. However, the inclusion of othercomponents in multiple copies renders them useful in other respects. Forinstance, the inclusion of a chelating agent containing a paramagneticion makes the emulsion useful as a magnetic resonance imaging contrastagent. The inclusion of biologically active materials makes them usefulas drug delivery systems. The inclusion of radionuclides makes themuseful either as therapeutic for radiation treatment or as diagnosticsfor imaging. Other imaging agents include fluorophores, such asfluorescein or dansyl. Biologically active agents may be included. Amultiplicity of such activities may be included; thus, images can beobtained of targeted tissues at the same time active substances aredelivered to them.

The emulsions can be prepared in a range of methods depending on thenature of the components to be included in the coating. In a typicalprocedure, used for illustrative purposes only, the following procedureis set forth: Perfluorooctylbromide (40% w/v, PFOB, 3M), and asurfactant co-mixture (2.0%, w/v) and glycerin (1.7%, w/v) is preparedwhere the surfactant co-mixture includes 64 mole % lecithin (PharmaciaInc), 35 mole % cholesterol (Sigma Chemical Co.) and 1 mole %dipalmitoyl-L-alpha-phosphatidyl-ethanolamine, Pierce Inc.) dissolved inchloroform. A drug is suspended in methanol (˜25 μg/20 μl) and added intitrated amounts between 0.01 and 5.0 mole % of the 2% surfactant layer,preferably between 0.2 and 2.0 mole %. The chloroform-lipid mixture isevaporated under reduced pressure, dried in a 50° C. vacuum ovenovernight and dispersed into water by sonication. The suspension istransferred into a blender cup (Dynamics Corporation of America) withperfluorooctylbromide in distilled or deionized water and emulsified for30 to 60 seconds. The emulsified mixture is transferred to aMicrofluidics emulsifier (Microfluidics Co.) and continuously processedat 20,000 PSI for three minutes. The completed emulsion is vialed,blanketed with nitrogen and sealed with stopper crimp seal until use. Acontrol emulsion can be prepared identically excluding the drug from thesurfactant commixture. Particle sizes are determined in triplicate at37° C. with a laser light scattering submicron particle size analyzer(Malvern Zetasizer 4, Malvern Instruments Ltd., Southborough, Mass.),which indicate tight and highly reproducible size distribution withaverage diameters less than 400 nm. Unincorporated drug can be removedby dialysis or ultrafiltration techniques. To provide the targetingligand, an α_(v)β₃ ligand is coupled covalently to the phosphatidylethanolamine through a bifunctional linker in the procedure describedabove.

Kits

The emulsions of the invention may be prepared and used directly in themethods of the invention, or the components of the emulsions may besupplied in the form of kits. The kits may comprise the pre-preparedtargeted composition containing all of the desired ancillary materialsin buffer or in lyophilized form. Alternatively, the kits may include aform of the emulsion which lacks the α_(v)β₃ ligand which is suppliedseparately. Under these circumstances, typically, the emulsion willcontain a reactive group, such as a maleimide group, which, when theemulsion is mixed with the targeting agent, effects the binding of thetargeting agent to the emulsion itself. A separate container may alsoprovide additional reagents useful in effecting the coupling.Alternatively, the emulsion may contain reactive groups which bind tolinkers coupled to the desired component to be supplied separately whichitself contains a reactive group. A wide variety of approaches toconstructing an appropriate kit may be envisioned. Individual componentswhich make up the ultimate emulsion may thus be supplied in separatecontainers, or the kit may simply contain reagents for combination withother materials which are provided separately from the kit itself.

A non-exhaustive list of combinations might include: emulsionpreparations that contain, in their lipid-surfactant layer, an ancillarycomponent such as a fluorophore or chelating agent and reactive moietiesfor coupling to the α_(v)β₃ targeting agent; the converse where theemulsion is coupled to targeting agent and contains reactive groups forcoupling to an ancillary material; emulsions which contain bothtargeting agent and a chelating agent but wherein the metal to bechelated is either supplied in the kit or independently provided by theuser; preparations of the nanoparticles comprising the surfactant/lipidlayer where the materials in the lipid layer contain different reactivegroups, one set of reactive groups for a α_(v)β₃ ligand and another setof reactive groups for an ancillary agent; preparation of emulsionscontaining any of the foregoing combinations where the reactive groupsare supplied by a linking agent.

Applications

The emulsions and kits for their preparation are useful in the methodsof the invention which include imaging of tissues containing highexpression levels of α_(v)β₃ integrin, and where tissues with suchexpression levels are undesirable, treatment. High expression levels ofα_(v)β₃ are typical of activated endothelial cells and are considereddiagnostic for neovasculature.

The diagnostic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 1 to 100 mCi per 70kg body weight, or preferably at a dose of 5 to 50 mCi. Imaging isperformed using known procedures.

The therapeutic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 0.01 to 5 mCi per kgbody weight, or preferably at a dose of 0.1 to 4 mCi per kg body weight.For comparable therapeutic radiopharmaceuticals, current clinicalpractice sets dosage ranges from 0.3 to 0.4 mCi/kg for Zevalin™ to 1-2mCi/kg for OctreoTher™, a labeled somatostatin peptide. For suchtherapeutic radiopharmaceuticals, there is a balance between tumor cellkill vs. normal organ toxicity, especially radiation nephritis. At theselevels, the balance generally favors the tumor cell effect. Thesedosages are higher than corresponding imaging isotopes.

The magnetic resonance imaging contrast agents of the present inventionmay be used in a similar manner as other MRI agents as described in U.S.Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt, et al., Magn.Reson. Med. (1986) 3:808; Runge, et al., Radiology (1988) 166:835; andBousquet, et al., Radiology (1988) 166:693. Other agents that may beemployed are those set forth in U.S. patent publication 2002/0127182which are pH sensitive and can change the contrast properties dependenton pulse. Generally, sterile aqueous solutions of the contrast agentsare administered to a patient intravenously in dosages ranging from 0.01to 1.0 mmoles per kg body weight.

A particularly preferred set of MRI chelating agents includes1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and itsderivatives, in particular, a methoxybenzyl derivative (DOTA-NCS)comprising an isothiocyanate functional group which can then be coupledto the amino group of phosphatidyl ethanolamine or to a peptidederivatized form thereof. Derivatives of this type are described in U.S.Pat. No. 5,573,752, incorporated herein by reference. Other suitablechelating agents are disclosed in U.S. Pat. No. 6,056,939, alsoincorporated herein by reference.

The DOTA isocyanate derivative can also be coupled to thelipid/surfactant directly or through a peptide spacer. The use ofgly-gly-gly as a spacer is illustrated in the reaction scheme below. Fordirect coupling, the DOTA-NCS is simply reacted with PE to obtain thecoupled product. When a peptide is employed, for example a triglycyllink, phosphoethanolamine (PE) is first coupled to t-boc protectedtriglycine. Standard coupling techniques, such as forming the activatedester of the free acid of the t-boc-triglycine using diisopropylcarbodiimide (or an equivalent thereof) with either N-hydroxysuccinimide (NHS) or hydroxybenzotriazole (HBT) are employed and thet-boc-triglycine-PE is purified.

Treatment of the t-boc-triglycine-PE with trifluoroacetic acid yieldstriglycine-PE, which is then reacted with excess DOTA-NCS in DMF/CHCl₃at 50° C. The final product is isolated by removing the solvent,followed by rinsing the remaining solid with excess water, to removeexcess solvent and any un-reacted or hydrolyzed DOTA-NCS.

For use as X-ray contrast agents, the compositions of the presentinvention should generally have a heavy atom concentration of 1 mM to 5M, preferably 0.1 M to 2 M. Dosages, administered by intravenousinjection, will typically range from 0.5 mmol/kg to 1.5 mmol/kg,preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is performed using knowntechniques, preferably X-ray computed tomography.

The ultrasound contrast agents of the present invention are administeredby intravenous injection in an amount of 10 to 30 μL of the echogenicgas per kg body weight or by infusion at a rate of approximately 3μL/kg/min. Imaging is performed using known techniques of sonography.

The methods of employing the nanoparticulate emulsions of the inventionare well known to those in the art. Typically, the tissues of interestto be imaged or treated include areas of inflammation, which maycharacterize a variety of disorders including rheumatoid arthritis,areas of irritation such as those affected by angioplasty resulting inrestenosis, tumors, and areas affected by atherosclerosis.

The following examples are offered to illustrate but not to limit theinvention.

Preparation a Part A—DSPE-PEG(2000)Maleimide-Mercaptoacetic Acid Adduct

1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Maleimide(PolyethyleneGlycol)2000] is dissolved in DMF and degassed by sparging with nitrogenor argon. The oxygen-free solution is adjusted to pH 7-8 using DIEA, andtreated with mercaptoacetic acid. Stirring is continued at ambienttemperatures until analysis indicates complete consumption of startingmaterials. The solution is used directly in the following reaction.

Part B—Conjugation of the DSPE-PEG(2000)Maleimide-Mercaptoacetic AcidAdduct With2-[({4-[3-(N-{2-[(2R)-2-((2R)-2-Amino-3-sulfopropyl)-3-sulfopropyl]ethyl}carbamoyl)propoxy]-2,6-dimethylphenyl}sulfonyl)amino](2S)-3-({7-[(imidazol-2-ylamino)methyl]-1-methyl-4-oxo(3-hydroquinolyl)}carbonylamino)propanoicAcid

The product solution of Part A, above, is pre-activated by the additionof HBTU and sufficient DIEA to maintain pH 8-9. To the solution is added2-[({4-[3-(N-{2-[(2R)-2-((2R)-2-amino-3-sulfopropyl)-3-sulfopropyl]ethyl}carbamoyl)propoxy]-2,6-dimethylphenyl}sulfonyl)amino](2S)-3-({7-[(imidazol-2-ylamino)methyl]-1-methyl-4-oxo(3-hydroquinolyl)}carbonylamino)propanoicacid, and the solution is stirred at room temperature under nitrogen for18 h. DMF is removed in vacuo and the crude product is purified bypreparative HPLC to obtain the Part B title compound.

EXAMPLE 1 Tumor Imaging

A. Tumor Model and Preparation of Nanoparticles:

Male New Zealand White Rabbits (˜2.0 kg) were anesthetized withintramuscular ketamine and xylazine (65 and 13 mg/kg, respectively). Theleft hind leg of each animal was shaved, sterile prepped and infiltratedlocally with Marcaine™ prior to placement of a small incision above thepopliteal fossa. A 2 by 2 by 2 mm³ Vx-2 carcinoma tumor fragment,freshly obtained from a donor animal, was implanted at a depth ofapproximately 0.5 cm. Anatomical planes were reapproximated and securedwith a single absorbable suture. Finally, the skin incision was sealedwith Dermabond skin glue. Following the tumor implantation procedure,the effects of xylazine were reversed with yohimbine and animals wereallowed to recover.

Twelve days after Vx-2 implantation rabbits were anesthetized with 1% to2% Isoflurane™, intubated, ventilated and positioned within the bore ofthe MRI scanner for study. Intravenous and intraarterial catheters,placed in opposite ears of each rabbit, were used for systemic injectionof nanoparticles and arterial blood sampling as described below. Animalswere monitored physiologically throughout the study in accordance with aprotocol and procedures approved by the Animal Studies Committee atWashington University Medical School.

At 12 days post-implantation, Vx-2 tumor volumes of animals receivingthe α_(v)β₃-targeted (130±39 mm³) or non-targeted nanoparticles (148±36mm³) were not different (p>0.05).

Twelve New Zealand rabbits implanted with Vx-2 tumors, as describedabove, were randomized into three treatment regimens and receivedeither:

1) α_(v)β₃-integrin-targeted paramagnetic nanoparticlesα_(v)β₃-targeted, n=4),

2) non-targeted paramagnetic nanoparticles (i.e., control group, n=4),or

3) α_(v)β₃-integrin-targeted non-paramagnetic nanoparticles followed byα_(v)β₃-integrin targeted paramagnetic nanoparticles (i.e., competitiongroup, n=4).

In treatment groups 1 and 2, rabbits received 0.5 ml/kg ofα_(v)β₃-integrin-targeted or control paramagnetic nanoparticlesfollowing the acquisition of baseline MR images. In treatment group 3,all rabbits received 0.5 ml/kg α_(v)β₃-integrin-targetednon-paramagnetic nanoparticles two hours before MR imaging followed by0.5 ml/kg α_(v)β₃-integrin-targeted paramagnetic nanoparticles. DynamicMR images were obtained at injection and every 30 minutes for eachanimal over two hours to monitor initial changes in signal enhancementin the tumor and muscle regions. All tumors were resected and frozen forhistology to corroborate MR molecular imaging results.

The paramagnetic nanoparticles were produced as described in Flacke, S.,et al., Circulation (2001) 104:1280-1285. Briefly, the nanoparticulateemulsions were comprised of 40% (v/v) perfluorooctylbromide (PFOB), 2%(w/v) of a surfactant co-mixture, 1.7% (w/v) glycerin and waterrepresenting the balance.

The surfactant of control, i.e., non-targeted, paramagnetic emulsionsincluded 60 mole % lecithin (Avanti Polar Lipids, Inc., Alabaster,Ala.), 8 mole % cholesterol (Sigma Chemical Co., St. Louis, Mo.), 2 mole% dipalmitoyl-phosphatidylethanolamine (DPPE) (Avanti Polar Lipids,Inc., Alabaster, Ala.) and 30 mole % gadoliniumdiethylenetriaminepentaacetic acid-bisoleate (Gd-DTPA-BOA, GatewayChemical Technologies, St. Louis, Mo.). The preparation of Gd-DTPA-BOAis described by Cacheris, W. P., et al., U.S. Pat. Nos. 5,571,498 and5,614,170, both incorporated herein by reference.

α_(v)β₃-targeted paramagnetic nanoparticles were prepared as above witha surfactant co-mixture that included: 60 mole % lecithin, 0.05 mole %N-[{w-[4-(p-maleimidophenyl)butanoyl]amino} poly(ethyleneglycol)2000]1,2-distearoyl-sn-glycero-3-phosphoethanolamine(MPB-PEG-DSPE) covalently coupled to the α_(v)β₃-integrin peptidomimeticantagonist (Bristol-Myers Squibb Medical Imaging, Inc., North Billerica,Mass.), 8 mole % cholesterol, 30 mole % Gd-DTPA-BOA and 1.95 mole %DPPE.

α_(v)β₃-targeted non-paramagnetic nanoparticles were prepared in anidentical fashion to the targeted formulation excluding the addition ofa lipophilic Gd chelate, which was substituted in the surfactantco-mixture with increased lecithin (70 mole %) and cholesterol (28 mole%).

The components for each nanoparticle formulation were emulsified in aM110S Microfluidics emulsifier (Microfluidics, Newton, Mass.) at 20,000PSI for four minutes. The completed emulsions were placed incrimp-sealed vials and blanketed with nitrogen.

Particle sizes were determined at 37° C. with a laser light scatteringsubmicron particle size analyzer (Malvern Instruments, Malvern,Worcestershire, UK) and the concentration of nanoparticles wascalculated from the nominal particle size (i.e., particle volume of asphere). The particle size distribution is shown in FIG. 1—most of theparticles had diameters less than 400 nm.

Perfluorocarbon concentration was determined with gas chromatographyusing flame ionization detection (Model 6890, Agilent Technologies,Inc., Wilmington, Del.). One ml of perfluorocarbon emulsion combinedwith 10% potassium hydroxide in ethanol and 2.0 ml of internal standard(0.1% octane in Freon®) was vigorously vortexed then continuouslyagitated on a shaker for 30 minutes. The lower extracted layer wasfiltered through a silica gel column and stored at 4-6° C. untilanalysis. Initial column temperature was 30° C. and ramped upward at 10°C./min to 145° C.

The gadolinium content of the emulsions was determined by neutronactivation analysis in a 300 kW nuclear reactor (Landsberger, S.,Chemical Analysis by Nuclear Methods, pp. 122-140, Z. B. Alfassi (ed.),New York: Wiley (1994)). The number of Gd complexes per nanoparticle wascalculated from the ratio of the concentrations of Gd³⁺ and theestimated number of nanoparticles in the emulsion. In addition, therelaxivities of each paramagnetic nanoparticle formulation were measuredat 0.47 Tesla and 40° C. with a Minispec Analyzer (Bruker, Inc., Milton,ON, Canada).

The characteristics of the particles are shown in Table 1.

Concentrations are reported relative to the total emulsion volume inliters. Relaxivity values (r₁ and r₂) were determined at 0.47 Tesla andcalculated relative to [Gd³⁺] or [nanoparticles] as indicated.

TABLE 1 Physical and Chemical Characteristics of α_(v)β₃-Targeted andNon-Targeted Nanoparticles α_(v)β₃-targeted Non-targeted Particle Size(nm) 273 263 Polydispersity Index 0.15 0.21 [Gd³⁺] (mM) 6.19 6.77 [¹⁹F](M) 28.9 28.6 [Particles] (nM) 65.5 73.3 Gd³⁺ Ions/Particle 94,40092,400 r₁ (s*mM)⁻¹ [Gd] 19.1 21.1 r₂ (s*mM)⁻¹ [Gd] 22.9 24.6 r₁ (s*mM)⁻¹[Particle] 1,800,000 1,950,000 r₂ (s*mM)⁻¹ [Particle] 2,160,0002,270,000

B. Magnetic Resonance Imaging and Histology Procedures

Twelve days after tumor implantation, the animals underwent MRI scanningon a 1.5 Tesla clinical scanner (NT Intera with Master Gradients,Philips Medical Systems, Best, Netherlands). Each animal was placedinside a quadrature head/neck birdcage coil with an 11 cm diametercircular surface coil positioned against the hindlimb near the tumor.The quadrature body coil was used for all radio-frequency transmission;the birdcage coil was used for detection during scout imaging; and thesurface coil was used for detection during high-resolution imaging. A 10ml syringe filled with gadolinium diethylenetriaminepentaacetic acid(Gd-DTPA) doped water was placed within the high-resolution field ofview (FOV) and served as a signal intensity standard.

Tumors were initially localized at the site of implantation with aT₂-weighted turbo spin-echo scan (TR: 2000 ms, TE: 100 ms, FOV: 150 mm,slice thickness: 3 mm, matrix: 128 by 256, signal averages: 2, turbofactor: 3, scan time: 3 min). A high-resolution, T₁-weighted, fatsuppressed, three-dimensional, gradient echo scan (TR: 40 ms, TE: 5.6ms, FOV: 64 mm, slice thickness: 0.5 mm, contiguous slices: 30, in-planeresolution: 250 μm, signal averages: 2, flip angle: 65°, scan time: 15min) of the tumor was collected at baseline and repeated immediately and30, 60, 90 and 120 minutes after paramagnetic nanoparticle injection.

Tumor volumes were calculated on an offline image processing workstation(EasyVision v5.1, Philips Medical Systems, Best, Netherlands).Regions-of-interest (ROI) were applied manually around the tumor in eachslice of the T₁-weighted baseline scan, were combined into athree-dimensional object and the volume calculated.

To quantify image enhancement over time, an unbiased image analysisprogram was used. T₁-weighted images (three contiguous slices throughthe center of each tumor) collected before, immediately after and 30,60, 90 and 120 minutes after intravenous nanoparticle injection wereanalyzed with MATLAB (The MathWorks, Inc., Natick, Mass.). The imageintensity at each timepoint was normalized to the baseline image via thereference gadolinium standard. Serial images were spatiallyco-registered and contrast enhancement was determined for each pixel ateach post-injection timepoint. An ROI was manually drawn around aportion of the hindlimb muscle in the baseline images and the averagepixel-by-pixel signal enhancement inside the ROI was calculated at eachtimepoint. A second ROI was manually drawn around the tumor and thestandard deviation of the tumor signal was calculated in the baselineimage for each animal. Pixels were considered enhanced when signalintensity was increased by greater than three times the standarddeviation of the tumor signal at baseline (i.e., enhancement greaterthan 99% of the variation seen at baseline). Solitary enhancing pixels,those in which all surrounding in-plane pixels did not enhance, wereremoved from the calculations as noise. The remaining enhancing pixelclusters were mapped back to the immediate, 30, 60 and 90 minute imagesand the average signal increase at each interval was determined.Statistical comparisons were performed for tumor and muscle for eachtimepoint using ANOVA (SAS, SAS Institute, Cary, N.C.). Treatment meanswere separated using the LSD procedure (p<0.05).

After imaging, tumors were resected for histology andimmunohistochemistry to verify tumor pathology and assess associatedvascularity and angiogenesis. Tumors were frozen (−78° C.) in OCT mediumwith known orientation relative to original anatomical position and theMRI image planes. Four micron frozen sections (Leica Microsystems, Inc.,Bannockburn, Ill.), fixed in acetone at −20° C. for 15 minutes and airdried overnight (4° C.), were stained with hematoxylin-eosin, murineanti-human/rabbit endothelium antibody (QBEND/40, 1:10 dilution,Research Diagnostics, Inc., Flanders, N.J.), or a murine anti-humanα_(v)β₃-integrin (LM-609, 1:200 dilution, Chemicon International,Temecula, Calif.). Immunohistochemistry was performed using theVectastain® Elite ABC kit (Vector Laboratories, Burlingame, Calif.94010), developed with the Vector VIP kit, counterstained with Vectormethylgreen nuclear counterstain. Slides were reviewed with a NikonEclipse E800 research microscope (Nikon USA, Melville, N.Y.) equippedwith a Nikon digital camera (Model DXM 1200) and captured with NikonACT-1 software.

C. Results of Imaging and Histology

T₁-weighted MR images of the Vx-2 tumor rabbits receivingα_(v)β₃-targeted paramagnetic nanoparticles revealed a marked increasein MR contrast primarily, although not exclusively, locatedasymmetrically along the tumor periphery. α_(v)β₃-integrin enhancementwas typically seen in a patchy distribution adjacent to blood vesselsand along tissue fascial interfaces (FIG. 2). Histology andimmunocytochemical assessments of the Vx-2 tumors corroborated thatangiogenesis was most intensely distributed within a few independentregions along the tumor periphery and found less extensively withinintratumoral connective tissue tracts interspersed between tumor celllobules (FIG. 3).

Temporally, MRI contrast enhancement provided by α_(v)β₃-targetedparamagnetic nanoparticles was detected in regions of angiogenesis soonafter injection at relatively low levels, which was presumablyattributed to a local extravasation of nanoparticles through afenestrated neovasculature at 30 minutes (FIG. 4). No intravascularblood pool contrast effect was detectable after 30 minutes. After twohours, the magnitude of signal enhancement among rabbits treated withthe α_(v)β₃-targeted nanoparticles increased (56%) relative to thenon-targeted nanoparticle effect (p<0.05). Blockage of α_(v)β₃-integrinsites with pretargeted non-paramagnetic α_(v)β₃-nanoparticles two hoursbefore injection of the α_(v)β₃-targeted paramagnetic particles reducedthe targeted contrast signal enhancement in half (p<0.05), to a signaleffect slightly below that attributed to localized neovascular leakage,confirming the specificity of the targeted nanoparticles.

In addition to the tumor capsule, contrast enhancement was shown in apatchy distribution among many of the vessels within the fossa and inparticular, within the wall of larger veins located only a fewmillimeters from capsular regions of angiogenesis. In one example, themagnitude of contrast signal enhancement determined for a venousangiogenic signal in close proximity to the tumor capsular signalincreased in parallel over time, suggesting a source and target (datanot shown). In many instances, angiogenesis stimulated in nearbyvasculature by factors elaborated from the tumor, clearly had notbridged to the tumor by 12 days post-implantation. Examination of thevasculature within the contralateral popliteal fossa revealed no MRsignal changes following injection with either α_(v)β₃-targeted ornon-targeted paramagnetic nanoparticles.

All Vx-2 rabbits routinely underwent baseline T₂-weighted MRI imaging atthe known site of surgery to localize tumor at 12 dayspost-implantation. In some rabbits, no tumor was detected and so thesewere excluded from the study. In a few other animals, a mass whichappeared appropriate based upon on size and T₂-image characteristics wasobserved, but later histology revealed it to be a tumor remnant withheavy infiltrates of inflammatory cells (FIG. 5); these animals wereexcluded from the study as well. The hyperintense appearance onT₂-weighted MRI is due to edema associated with inflammation.

Among this subset of animals, some randomly received α_(v)β₃-targetedparamagnetic nanoparticles and no MR contrast enhancement was shownwithin the periphery of the mass nor within nearby vasculature (FIG. 6).This lack of signal enhancement associated with a popliteal mass oradjacent vasculature was distinct from the molecular imaging featuresroutinely obtained in animals with histologically verified tumor.Histology and immunohistochemical analysis of the remnant tissuesconfirmed a paucity of vascularity in the tumor periphery and adjacenttissues with negligible staining for the α_(v)β₃-integrin. Thesefindings illustrate the specificity of molecular imaging to helpdifferentiate viable Vx-2 masses from tumor remnants.

EXAMPLE 2 Imaging of Atherosclerosis

A. Model System and Nanoparticles

Both targeted and non-targeted nanoparticles were prepared as describedin paragraph A of Example 1. The characteristics obtained weresimilar—the particles contained 6.17 mM Gd, or about 94,200 Gdatoms/particle. The nominal particle size measured by elastic lightscattering (Malvern Instruments, Worchestershire, UK) was 273 nm with a“polydispersity index” (or distribution bandwidth) of 0.15.

The actual T1 and T2 relaxivities (r1 and r2, respectively) of theparticle formulations were determined with the use of standard inversionrecovery pulse sequences and multiecho sequences applied to pure samples(nanoparticles present at 59 nM) placed in a quadrature birdcage coiland imaged with a clinical 1.5 T system (Philips NT Intera CV, PhilipsMedical Systems, Best, Netherlands). The “ionic-based” r1 and r2 valuesfor paramagnetic nanoparticles expressed per mM Gd³⁺ are 17.7±0.2 and25.3±0.6 (sec·mM)¹ respectively. “Particle-based” relaxivities are1,670,000±100,000 and 2,380,000±120,000 (sec·mM particle)⁻¹ for r1 andr2. These relaxivities are more than 5 orders of magnitude greater thanthose for commercially available paramagnetic contrast agents.

The targeted nanoparticles each contained approximately 200-300 copiesof the peptidomimetic linked to the particle lipid membrane through thecoupled phospholipid, described in Preparation A, part B. Physicalcharacteristics of the nanoparticles were unaffected by the inclusion ofthe targeting ligand, including pharmacokinetic properties, and bothtargeted and control particles exhibited indistinguishable paramagneticproperties.

To induce atherosclerosis, 13 male New Zealand White rabbits were fedeither 1% cholesterol (n=9) or standard rabbit chow (n=4) for ˜80 days.The contrast agents were injected intravenously via ear vein in a doseof 0.5 ml/kg body weight; i.e., about 10¹⁴ nanoparticles per dose. Threeexperimental groups were used:

1) Control diet animals administered α_(v)β₃-targeted paramagneticnanoparticles (n=4);

2) High-cholesterol rabbits administered α_(v)β₃-targeted nanoparticles(n=5) or

3) High-cholesterol rabbits administered non-targeted controlnanoparticles (n=4).

Following MRI, all aortas extracted for histological assessment. Routinehematoxylin/eosin staining was performed on formalin-fixed, paraffinembedded sections (4?m) of aorta. Expression of α_(v)β₃ integrin in theaortic wall was confirmed by immunohistochemistry of formalin-fixedsections with use of a specific primary antibody (LM609: ChemiconInternational, Inc., Temecula, Calif.), and secondary antibody developedwith VIP substrate Kit. PECAM was stained similarly with CD31 primaryantibody (Chemicon International, Inc., Temecula, Calif.). Images ofneovasculature were digitized under high power (600×) with a Nikonmicroscope and Nikon DXM1200 camera.

The experimental protocol was approved by the Animal Studies Committeeof the Washington University School of Medicine.

B. Imaging and Histology

MR images were obtained in a manner similar to that set forth inparagraph B of Example 1. A 1.5 T magnet (NT Intera CV, Philips MedicalSystems, Best, Netherlands) was used along with a quadrature birdcage RFreceive coil to image the aorta in vivo before and after treatment withparamagnetic nanoparticles. Multislice T1-weighted spin-echo,fat-saturated, black-blood imaging of the aorta was performed from therenal arteries to the diaphragm (TR 380 ms; TE 11 ms; 250 by 250 μmin-plane resolution, 5 mm slice thickness; NSA=8). Although the actualTR used for imaging in vivo was not optimal, according to our signalsimulations, it provided a practical means to acquire the data in ashort period of time. The effect on signal intensity is best illustratedas having to approximately double the nanoparticle concentration (toaround 100 μM) to achieve a CNR=5 at 1.5 T. To null the blood signal,“sliding rf” saturation bands were placed proximal and distal to theregion of image acquisition and moved with the selected imaging plane.

C. Results of Imaging and Histochemistry

The use of targeted nanoparticles showed enhancement of the contrastimage in locations verified as associated with atherosclerosis.

FIG. 7A (top) shows the imaged portion of the aorta in longitudinalprofile for a selected animal and transverse slices (bottom) before and120 minutes after treatment with targeted nanoparticles, and also anexample of output from a custom-designed image segmentation algorithmfor quantitative signal analysis of individual aortic slices. The signalin the aortic wall is increased after contrast injection (middle panel),indicating the presence of targeted nanoparticles that have bound to theα_(v)β₃ integrin epitopes. Furthermore, the aortic blood pool backgroundis not confounding (note: low blood signal in lumen) in view of thesmall doses of nanoparticles used and the “black blood” signal nullingprocedure, which enables immediate detection of contrast enhancement inthe aortic wall without requiring a waiting period for blood poolclearance of contrast agent.

FIG. 7B shows the variation of contrast enhancement longitudinally alongthe aorta for three selected rabbits. Overall, greater signalenhancement was observed in the high-cholesterol targeted rabbits atpractically all aortic segments. As shown, the percent enhancement withtargeted particles in the rabbit fed a high cholesterol diet wasmarkedly higher than either the enhancement of image using non-targetednanoparticles in a rabbit fed a high cholesterol diet (open squares) andhigher than the enhancement using targeted particles in a rabbit fed anormal diet (solid triangles).

Variability of contrast enhancement within the aortic wall wasdetermined for three rabbits 120 minutes after treatment and showedsignificant signal heterogeneity at individual aortic levels. The highcholesterol rabbit administered targeted nanoparticles in particularmanifests the greater overall enhancement, but “hot spots” are presentin all three samples.

Histological determinations confirmed the colocalization of the α_(v)β₃integrin epitopes with the vascular endothelium. H&E staining showedthat there was mild intimal thickening after 80 days in thecholesterol-fed rabbits only. Immunocytochemical analyses revealedprominent staining for α_(v)β₃ at the adventitia-media interface in thecholesterol-fed rabbits and PECAM staining, indicative of vascularendothelium, colocalized with the α_(v)β₃ integrin epitopes at theadventitia-media interface. This was observed much more prominently inthe cholesterol-fed rabbits, confirming the presence of an expanded vasavasorum associated with inflammatory markers.

A “region-growing” segmentation algorithm for semi-automated analysis ofsignal intensities within the aortic wall images for each imaged slicewas developed. The aortic lumen was isolated in each two-dimensionalimage through the use of a seeded “region-growing” algorithm thatiteratively increased the segmented area by evaluating the surroundingpixels for their similarity to the previously segmented pixels. Once apre-determined threshold for similarity was reached, growth terminated.By increasing the width of this segmentation to include the wall andsubtracting the previously segmented lumen, only a binary mask of theaortic wall and some additional background pixels remained. Furtherthresholding was used to remove the background pixels so that only theaortic wall was segmented. After segmentation, the mean intensity of thewall in each slice and time point was subtracted from the mean intensityin the same slice at baseline. The algorithm kernel was adapted from aprocedure developed by Dr. Michael Brown at the Hong Kong University ofScience and Technology available at a www address of cs.ust.hk/˜brown/.

This procedure was applied uniformly to all aortic data sets andproduces a circumferential region of interest for the entire aortic wallas illustrated in FIG. 7A. MRI signal intensity before and afternanoparticle injection was quantified within the entire segmented aorticregion at each level, and in adjacent skeletal muscle regions ofinterest that were selected at random. Signal intensity was normalizedto the signal from a fiduciary marker (a Gd³⁺-DTPA/saline solution in atest tube phantom) that was placed within the field of view. The percentchange in signal intensity after nanoparticle injection was calculatedfor images at 15, 60, and 120 minutes after injection. General linearmodeling with Duncan's multiple-range testing of group differences (SAS,Inc., Cary, N.C.) was used to determine the significance of differencesin MRI signals (p<0.05).

Quantification of the aortic signal enhancement was conductedconservatively by calculating the average aortic enhancement for asingle rabbit across all aortic levels that had been imaged, and thenaveraging these single rabbit values for an entire experimental group.FIG. 8A shows that immediately after injection of targeted nanoparticles(within approximately 15 minutes), the signal in the entire aortic wallwas enhanced by 26±3.8% for all rabbits. By 120 minutes, the signal fromthe entire aortic wall was enhanced further by 47±5.4% over baseline.The entire aortic wall of all cholesterol-fed rabbits that receivednon-targeted nanoparticles also enhanced by 19±0.8% within 15 minutes,but remained stable from 60-120 minutes (26±1%), which represents onlyabout half of the signal augmentation as for the specifically targetedenhancement.

In the control-diet rabbits, significant aortic wall enhancement wasobserved immediately after injection of targeted nanoparticles to alevel equivalent to that of the cholesterol-fed rabbits at that timepoint (14.5±2.2%). However, after 2 hours, the signal was littleincreased (23.7±3.7%). Thus, the signal enhancement in the entire aorticwall for cholesterol-fed animals approximately doubled that forcontrol-diet animals by 120 minutes.

The signal enhancement observed in the adjacent skeletal muscle (FIG.8B) for any group at any time period was far less than that for any ofthe aorta groups, and just bordered statistical significance (p<0.051).This trend was not related statistically to nanoparticle type or tofeeding regimen by ANOVA.

The data indicate that specific identification of α_(v)β₃ epitopes invascular inflammation is possible with high resolution MRI in vivo.

Previous pharmacokinetic analyses indicate that particle clearance isbiexponential with a β-elimination rate of 1-1.5 hours. These propertiesare not affected by addition of the ligand or the gadolinium chelate.Accordingly, the concentration gradient driving nonspecific accumulationof molecularly-targeted nanoparticles in aorta or muscle should bediminishing by 120 minutes (which is consistent with the present datashowing a plateau for nonspecific signal enhancement in aorta: see FIG.8). On the contrary, the process of specific binding to α_(v)β₃ epitopesshould increase for a number of half lives since the nanosystem isexpected to exist in large ligand excess in the circulation as comparedwith the low prevalence of molecular epitopes on the neovasculature,given that approximately 100 trillion nanoparticles were injected in anaverage i.v. dose. The greater nonspecific enhancement in aortic wallversus that for muscle likely relates to the expansion of thesinusoid-like vasa vasorum that provides both a larger distributionvolume, and simultaneously a greater local concentration of paramagneticnanoparticles that are less subject to signal nulling by our particular“black blood” imaging method. Because inflow/outflow in the vasa vasorumis likely to be much slower than that in the aortic lumen, signalnulling should be more effective for the aortic lumen blood pool thanfor that of the vasa vasorum.

Targeted paramagnetic nanoparticles can thus be used with MRI in smalli.v. doses with routine clinical imaging approaches to delineatevascular inflammation and/or angiogenesis in early stageatherosclerosis.

EXAMPLE 3 Restenosis Model

Domestic pigs, healthy, diabetic, or hyperlipidemic, are sedated withtelazol cocktail (1 ml/23 kg IM) followed by intubation and 1-2%isoflurane anesthesia in oxygen. The ECG, blood gases and arterial bloodpressure are monitored. Lidocaine, diltiazem, and/or nitroglycerin areused to treat vasospasm.

Following peripheral arterial access and sheath placement, anappropriate sized angioplasty balloon, e.g., 8 mm×2 cm balloon catheter;Proflex, Mallinckrodt Inc., St. Louis), is positioned at a cervicalvertebral level (C-3 to C-5) and inflated multiple times (usually 3times) to a pressure of 6 atmospheres for 30 seconds with 60 secondpauses between inflations. A balloon-to-artery ratio of approximately1.5 is typically employed. This procedure produces a consistent ruptureof the internal elastic lamina and injury to the media.

Following the above carotid overstretch balloon-injury, an emulsioncomprising nanoparticles, as described in Example 1, is administered viaa local delivery catheter system. The delivery system is a pairedballoon catheter or an mechanical perfusion delivery/vacuum extractionsystem. Targeted- or control nanoparticles, or saline alone aredelivered locally and allowed to incubate for between 1 and 15 minutes.An MR angiogram is performed prior to carotid vascular wall imagingstudies.

MRI scanning is performed on a 1.5 Tesla clinical scanner (NT Intera CV,Philips Medical Systems, Best, Netherlands) or comparable clinicalsystem at 1.0T to 7.0T. Appropriate coils include a quadrature head/neckbirdcage coil, circular surface coils, phased-array (Synergy) coils. Forresearch analyses, gadolinium diethylenetriaminepentaacetic acid(Gd-DTPA) doped water standards are placed within the high-resolutionfield of view (FOV) to serve as an image signal intensity standard; thisis not required for clinical application. MR image analysis is performedoff-line with an EasyVision v5.1 workstation (Philips Medical Systems,Best, Netherlands) or similar image manipulation system.

FIG. 9 shows a 3-dimensional reconstruction of the contrast-enhancedballoon injury pattern using α_(v)β₃-targeted paramagneticnanoparticles. This reveals the spatial distribution of microfracturesinduced within the tunica media. These data, impossible to detect withroutine X-ray angiography, can provide quantitative assessments of wallinjury that have prognostic importance to subsequent revascularizationcomplications, including restenosis.

In addition to comprising the α_(v)β₃ targeting moiety, thenanoparticles are supplied with antiproliferative agents such asradionuclides, paclitaxel or rapamycin.

1. A composition comprising an emulsion of nanoparticles, wherein saidnanoparticles consist of liquid perfluorocarbon cores coated withlipid/surfactant, and wherein said nanoparticles are coupled to a ligandspecific for α_(v)β₃, which comprises the formula

and wherein the coupling of said nanoparticles to the ligand is througha covalent linkage, through a spacer, to a component of thelipid/surfactant coating, and wherein said nanoparticles incorporate atleast one imaging agent.
 2. The composition of claim 1, wherein saidresidue is coupled through the spacer to a component of thelipid/surfactant coating that is a phosphatidyl lipid.
 3. Thecomposition of claim 2, wherein the ligand coupled to the component ofthe lipid/surfactant coating is of the formula


4. The composition of claim 1, wherein said imaging agent is aradioisotope.
 5. The composition of claim 1, wherein the imaging agentis a fluorophore.
 6. The composition of claim 1, wherein the imagingagent is a magnetic resonance imaging (MRI) contrast agent.
 7. Thecomposition of claim 6, wherein said MRI contrast agent is a chelatedparamagnetic ion.
 8. The composition of claim 7, wherein theparamagnetic ion is gadolinium ion.
 9. The composition of claim 1, whichfurther contains a biologically active agent.
 10. The composition ofclaim 10, wherein said biologically active agent is a hormone orpharmaceutical.
 11. The composition of claim 10, wherein thepharmaceutical compound is an antineoplastic agent, an analgesic, ananesthetic, a neuromuscular blocker, an antimicrobial agent, anantiparasitic agent, an antiviral agent, an interferon, an antidiabetic,an antihistamine, an antiitussive, or an anticoagulant, or anantiproliferative.
 12. The composition of claim 11, wherein thepharmaceutical compound is an antiproliferative.
 13. The composition ofclaim 12, wherein the pharmaceutical compound is paclitaxel orrapamycin.